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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/44—Polycarboxylic acids
  • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/42—Hydroxy-carboxylic acids
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to a method of carrying out electromicrobial reductions with the aid of microorganisms which receive the reduction equivalents via an electron carrier which is regenerated electrochemically.
• Electrons e are transferred to an electron carrier EC ox which passes them on to a reductase R which reduces, for example, oxidized nicotinamide adenine dinucleotide (NAD.sup. ⁇ ⁇ NADH); cf. J. Elektroanal. Chem. 32 (1971), 415 and J. Org. Chem. 46 (1981), 4623).
• NADH oxidized nicotinamide adenine dinucleotide
• E oxidized nicotinamide adenine dinucleotide
• the costs of enzyme concentration and the instability of pure enzymes prevent the use of these methods for preparative purposes. Attempts have therefore been made to carry out the reduction using whole anaerobic cells which contain reductase (Angew. Chem. 93 (1981), 897). This process, too, is scarcely more suitable for large-scale syntheses, because anaerobic microorganisms are sensitive to oxygen, and their growth and
• the present invention relates to a method of carrying out electromicrobial reductions with the aid of microorganisms which receive the reduction equivalents via an electron carrier which is regenerated electrochemically, wherein the microorganisms employed are aerobic or microaerophilic ones, and the reduction is carried out in the absence of oxygen.
• the novel electromicrobial reduction takes place in accordance with the following equation: ##STR2##
• the electrons e originate from a current source and are transferred to an electron carrier EC ox , which passes them on to the aerobic microorganism M ox .
• This microorganism transfers the electrons together with two protons to the substrate S, which is reduced to SH 2 .
• the reduction proceeds very smoothly.
• microorganisms which contain NADH-dependent or NADPH-dependent reductases, including microorganisms which become permeable to NADH or NADPH under the reaction conditions and slowly lose these compounds.
• microorganisms which contain enzymes which slowly decompose these pyridine nucleotides also exist. In the last two cases, it is advisable to accelerate the reaction by adding a small amount of a pyridine nucleotide to the reaction mixture.
• the reduction is preferably carried out in a compartmented cell at from 5° to 90° C., advantageously from 10° to 50° C., preferably from 20° to 40° C., and at a pH of from 3 to 10, preferably from 5 to 8.
• the electrodes are produced from electrode material conventionally used in electrosynthesis.
• cathodes consisting of a metal, eg. lead, copper, iron, nickel, mercury or a steel or graphite semiconductor, or of Nafion doped with a viologen dye
• anodes consisting of platinum or graphite or dimensionally stable anodes of doped or coated titanium, as used for the production of oxygen or chlorine, are suitable.
• the partition between the anolyte and the catholyte is a commercial diaphragm or membrane, preferably an ion-exchange membrane, as used, for example, for chlor-alkali electrolysis or for electrodialysis.
• the current density is from 1 to 200, preferably from 1 to 100, mA/cm 2
• the cathode potential is from -0.1 to -1.5 V, preferably from -0.5 to -0.9 V, with reference to standard calomel electrodes
• the terminal voltage of the cell is from 2 to 90 V, preferably from 4 to 20 V.
• the electrolysis is carried out as a rule in an aqueous mixture which, in addition to the microbial system and the substrate, can also contain conductive salts, buffers and organic solvents or solubilizers, for example alcohols, such as methanol or ethanol, ethers, such as dioxane, dimethoxyethane or methyl tert.-butyl ether, emulsifiers, such as polyoxyethylene sorbitan monooleate, esters, such as ethyl acetate, alkanes, such as hexane or petroleum ether, chlorohydrocarbons, such as methylene chloride, carbon tetrachloride or chloroform, or dimethylformamide.
• conductive salts for example alcohols, such as methanol or ethanol, ethers, such as dioxane, dimethoxyethane or methyl tert.-butyl ether, emulsifiers, such as polyoxyethylene sorbitan
• Organic solvents may be used, particularly in combination with immobilized cells.
• solvents are saturated alcohols, dioxane, furan, dimethylsulfoxide, etc.
• the procedure may be carried out in a multi-phase system, one phase comprising a hydrocarbon, ether or higher alcohol.
• an organic solvent can be advantageous if this allows a heterogeneous reaction procedure (eg. solid/liquid) to be avoided.
• a heterogeneous reaction procedure eg. solid/liquid
• the reaction results in a product which is soluble in organic solvents and which attacks by the microorganism or the enzyme present in this, it may be appropriate to carry out the procedure using a 2-phase system.
• the anolyte consists of an aqueous salt solution, examples of suitable salts for this solution being NaCl, Na 2 SO 4 and NaO--CO--CH 3 .
• the salt solution it is also possible to use a dilute aqueous mineral acid.
• the catholyte also consists of a salt solution, which additionally contains the substrate and the microorganism.
• a buffer such as a phosphate buffer, is advantageous.
• Suitable electron carriers are:
• Viologen dyes eg. methyl viologen, benzyl viologen and diquat
• anthraquinone and other quinone dyes eg. phenosafranine, methylene blue and 2-anthraquinonesulfonic acid
• triphenylmethane dyes eg. methyl violet and crystal violet
• phthalocyanines eg. Fe phthalocyanine, Cu phthalocyanine and Co phthalocyanine
• methine dyes eg. astraphloxin
• imidazole derivatives eg. metronidazole
• thiols eg. dihydroliponic acid, dithiothreitol, 2-mercaptoethanol, glutathione, thiophenol and butane-1,4-dithiol, and
• the 1st group is preferred, and methyl viologen and benzyl viologen are particularly preferred.
• Suitable microorganisms for the reduction reaction are all aerobic microorganisms which contain the enzymes required for the desired reaction.
• Important examples of microorganisms are:
• Gram-negative aerobic bacteria eg. Acetobacter ascendens, Acetobacter pasteurianus, Alcaligenes eutrophus, Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas testosteroni, Gram-negative facultatively anaerobic bacteria, eg. Enterobacter aerogenes, Enterobacter agglomerans, Escherichia coli, Flavobacterium spec., Proteus mirabilis, Proteus vulgaris, Proteus mitajiri and Zymomonas mobilis, Gram-positive cocci, eg.
• Leuconostoc mesenteroides Peptococcus aerogenes, Sarcina lutea and Streptococcus faecalis, endospore-forming bacteria, eg. Bacillus subtilis, Bacillus cereus and Bacillus polymyxa, Gram-positive asporogenic bacteria, eg. Lactobacillus buchneri, coryneform bacteria, eg. Arthrobacter spec. and Corynebacterium simplex, and actinomycetes, eg. Actinomyces globosus, Mycobacterium spec., Nocardia corallina, Streptomyces platensis and Streptomyces lavendulae;
• Phycomycetes eg. Absidia orchidis, Rhizopus arrhizus, Rhizopus nigricans and Rhizopus reflexus, protoascomycetes (yeasts), eg. Candida pseudotropicalis, Geotrichum candidum, Hansenula capsulata, Kloeckera magna, Kluyveromyces fragilis, Rhodotorula mucilaginosa, Rhodotorula glutinis, Saccharomyces cerevisiae, Saccharomyces sake, Saccharomyces fragilis, Saccharomyces uvarum, Schizosaccharomyces pombe, Candida utilis and Candida boidenii, ascomycetes, eg.
• microorganisms are also aerobic protozoa and aerobic cells of higher plants and animals, provided that these can be grown like microorganisms. Such microorganisms can, for example, be obtained from depositories or be self-grown.
• Proteus mirabilis Proteus vulgaris, Alcaligenes eutrophus, Bacillus cereus, Geotrichum candidum, Kloeckera magna, Saccharomyces cerevisiae and Candida utilis are particularly preferred.
• one microorganism is particularly effective in producing NADH or NADPH, while the reductase has a high activity with respect to the conversion of the substrate S in another microorganism, but the latter microorganism has only a low activity with regard to the formation of NADH or NADPH. In these cases, it is advisable to use a mixture of the two microorganisms.
• microorganisms or cells can also be used in immobilized form for the conversions. Furthermore, the permeability of the microorganisms to cosubstrates, substrates and products can be increased in a number of cases, for example by freezing and thawing out the cells.
• oxygen content must be sufficiently low that any reaction between the oxygen and the electron carrier which may take place is unimportant, and that the oxygen or an oxidation product formed as a result of the presence of oxygen has no inhibitory effect on the enzymes and cosubstrates present during the reaction. If the oxygen content of the catholyte increases, for example, where methyl viologen is used, to above 5.10 -7 M, the current efficiency and the stability of the biocatalysts decrease with increasing oxygen content.
• Y is COO--, CHO or COR
• X is H, alkyl, alkoxy, alkylthio, halogen, dialkylamino or arylamino
• R 1 and R 2 are each H, alkyl, alkoxy, aryl, alkoxycarbonyl or alkenyl.
• Such hydrogenation products are chiral halocarboxylic acids, eg. 3-(p-chlorophenyl)-2-chloropropionic acid, chiral ⁇ - and ⁇ -alkyl-branched carboxylic acids, eg. (R)- and (S)-2-methyl-3-phenylpropionic acid and 2-amino-3-methyl-3-phenylpropionic acid, and ⁇ 3 -2- and/or 4-substituted carboxylic acids obtained from the corresponding allenecarboxylic acids.
• a racemate molecular asymmetry
• Examples of the reduction of aldehydes to chiral products include the preparation of (R)- or (S)-citronellal or citronellol from cis- or trans-citral.
• the end product is isolated from the reaction solution in a conventional manner, for example by distillation, extraction, crystallization or chromatography.
• the novel method Compared with methods carried out using anaerobic microorganisms, the novel method has the following advantages:
• Aerobic microorganisms are insensitive to oxygen and hence much easier to use.
• Aerobic microorganisms are simpler to produce than anaerobic ones. Moreover, they give substantially higher cell densities, so that less expense is entailed with regard to the apparatus required to produce them.
• methyl viologen 50 ⁇ mole of methyl viologen, 2.5 millimoles of potassium phosphate and 400 mg of Candida utilis (eg. DSM 70,167) were dissolved or suspended in 25 ml of water, and the pH was brought to 7.0. The resulting mixture was introduced into an electrochemical cell, and the methyl viologen was reduced at a constant cathode potential of -790 mV with reference to a standard calomel electrode (SCE). The zero current was about 0.25 mA. Thereafter, 1 millimole of acetol was added, and the potential was maintained at -790 mV.
• SCE standard calomel electrode
• the reduction could also be carried out using Proteus mirabilis (DSM 30,115) and Proteus vulgaris (DSM 30,118). In these cases, a current of 15 mA per 20 mg of cell material was obtained.
• Example 5 The crude lysates of the two microorganisms mentioned in Example 5 and the other components, as well as the phenylpyruvate or 2-oxo-4-methylpentanate, were converted in an electrochemical cell by a procedure similar to that described in Example 5. During the reduction, the current was about 0.5 mA.

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• 1982
  • 1982-07-17 DE DE19823226888 patent/DE3226888A1/de not_active Withdrawn
• 1983
  • 1983-07-06 DE DE8383106608T patent/DE3367935D1/de not_active Expired
  • 1983-07-06 AT AT83106608T patent/ATE23879T1/de not_active IP Right Cessation
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